Conservation of human Y chromosome sequences among male great apes: Implications for the evolution of Y chromosomes

Nine newly described single-copy and lowcopy-number genomic DNA sequences isolated from a flow-sorted human Y chromosome library were mapped to regions of the human Y chromosome and were hybridized to Southern blots of male and female great ape genomic DNAs (Gorilla gorilla, Pan troglodytes, Pongo pygmaeus). Eight of the nine sequences mapped to the euchromatic Y long arm (Yq) in humans, and the ninth mapped to the short arm or pericentromeric region. All nine of the newly identified sequences and two additional human Yq sequences hybridized to restriction fragments in male but not female genomic DNA from the great apes, indicating Y chromosome localization. Seven of these 11 human Yq sequences hybridized to similarly-sized restriction endonuclease fragments in all the great ape species analyzed. The five human sequences that mapped to the most distal subregion of Yq (deletion of which region is associated with spermatogenic failure in humans) were hybridized to Southern blots generated by pulsed-field gel electrophoresis. These sequences define a region of approximately 1 Mb on human Yq in which HpaII tiny fragment (HTF) islands appear to be absent. The conservation of these human Yq sequences on great ape Y chromosomes indicates a greater stability in this region of the Y than has been previously described for most anonymous human Y chromosomal sequences. The stability of these sequences on great ape Y chromosomes seems remarkable given that this region of the Y does not undergo meiotic recombination and the sequences do not appear to encode genes for which positive selection might occur. Correspondence to: B. Steele Allen     

Comparative mapping of ZFY in the hominoid apes

Summary Within our project of comparative mapping of candidate genes for sex-determination/testis differentiation, we used a cloned probe from the human ZFY locus for comparative hybridization studies in hominoids. As in the human, the ZFY probe detects X- and Y-specific restriction fragments in the chimpanzee, the gorilla, the orangutan, and the gibbon. Furthermore, the X-specific hybridization site in the great apes resides in Xp21.3, the same locus defining ZFX in the human. The Y-specific locus of ZFY maps closely to the early replicating pseudoautosomal segment in the telomeric or subtelomeric position of the Y chromosomes of the great apes, again as found in the human. Thus, despite cytogenetically visible structural alterations within the euchromatic parts of the Y chromosomes of the human species and the great apes, a segment of the Y chromosome defined by the pseudoautosomal region and ZFY seems to be more strongly conserved than the rest of the Y chromosome.     

Characterization and evolution of a single-copy sequence from the human Y chromosome.

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.     

Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.     

A human moderately repeated Y-specific DNA sequence is evolutionarily conserved in the Y chromosome of the great apes.

Evolutionary conservation of the human-derived moderately repeated Y-specific DNA sequence Y-190 (DYZ5) was investigated in the chimpanzee, orangutan, and gorilla. Southern blot analysis showed the presence of the sequence in the Y chromosome of all great apes. Pulsed-field gel electrophoresis and in situ hybridization revealed that the repeat is organized in one major block and confined to a small region of the Y chromosome of the three species. DYZ5 was assigned to the proximal short arm of the Y chromosome of the chimpanzee and orangutan and to the long arm of the Y chromosome of the gorilla. In light of its evolutionary conservation, DYZ5 may have an as yet undetermined structural function in the Y chromosome.     

Construction, analysis, and application to 46,XY gonadal dysgenesis of a recombinant phage DNA library from flow-sorted human Y chromosomes

The analysis of a recombinant human Y-enriched Hind III total digest phage library prepared from the DNA of flow sorted human Y chromosomes is described. Out of 43 phage inserts from the library thus far mapped, 25 revealed hybridization with Y chromosomal DNA. These inserts may be divided into five groups according to their degree of Y specific hybridization: inserts that hybridize with one single copy or slightly repeated Y-specific DNA sequence, Y-specific repeated sequences of various restriction fragment lengths, Y-chromosomal DNA sequence(s) shared by a sequence on the X and/or on autosomes, Y-specific DNA sequences in addition to multiple X and/or autosomal sequences, or Y-specific repeated DNA in addition to multiple X and/or autosomal sequences. Application of probes from this library for diagnostic purposes is shown in two 46,XY patients with gonadal dysgenesis and small deletions of the Y short arm.     

Concerted evolution of primate alpha satellite DNA. Evidence for an ancestral sequence shared by gorilla and human X chromosome alpha satellite

To understand evolutionary events in the formation of higher-order repeat units in alpha satellite DNA, we have examined gorilla sequences homologous to human X chromosome alpha satellite. In humans, alpha satellite on the X chromosome is organized as a tandemly repeated, 2.0 x 10(3) base-pairs (bp) higher-order repeat unit, operationally defined by the restriction enzyme BamHI. Each higher-order repeat unit is composed of 12 tandem approximately 171 base-pair monomer units that have been classified into five distinct sequence homology groups. BamHI-digested gorilla genomic DNA hybridized with the cloned human 2 x 10(3) bp X alpha satellite repeat reveals three bands of sizes approximately 3.2 x 10(3), 2.7 x 10(3) and 2 x 10(3) bp. Multiple copies of all three repeat lengths have been isolated and mapped to the centromeric region of the gorilla X chromosome by fluorescence in situ hybridization. Long-range restriction mapping using pulsed-field gel electrophoresis shows that the 2.7 x 10(3) and 3.2 x 10(3) bp repeat arrays exist as separate but likely neighboring arrays on the gorilla X, each ranging in size from approximately 200 x 10(3) to 500 x 10(3) bp, considerably smaller than the approximately 2000 x 10(3) to 4000 x 10(3) bp array found on human X chromosomes. Nucleotide sequence analysis has revealed that monomers within all three gorilla repeat units can be classified into the same five sequence homology groups as monomers located within the higher-order repeat unit on the human X chromosome, suggesting that the formation of the five distinct monomer types predates the divergence of the lineages of contemporary humans and gorillas. The order of 12 monomers within the 2 x 10(3) and 2.7 x 10(3) bp repeat units from the gorilla X chromosome is identical with that of the 2 x 10(3) bp repeat unit from the human X chromosome, suggesting an ancestral linear arrangement and supporting hypotheses about events largely restricted to single chromosome types in the formation of alpha satellite higher-order repeat units.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Transposition de la Séquence CB1 du Chromosome X des Singes Anthropoïdes à I'Y Humain

The genomic probe CB1 recognizes sites of simple sequence homotogy between the human X and Y chromosomes. DNAs restricted with EcoRI from the great apes (Pan and Gorilla) were examined for sequences homologous to this probe, by molecular hybridization experiments. Results reported here revealed, by genetic dosage with sex and using an internal standard, that the great apes have homologous sequences of this probe only on their X chromosome. Consequently, the corresponding sequences were transposed from the X to the Y chromosomes after the human line diverged. Comparative patterns of the sequence restricted by 10 restriction enzymes between woman and man show that the transposition is of recent origin.     

Evolution of homologous sequences on the human X and Y chromosomes, outside of the meiotic pairing segment.

A sequence isolated from the long arm of the human Y chromosome detects a highly homologous locus on the X. This homology extends over at least 50 kb of DNA and is postulated to be the result of a transposition event between the X and Y chromosomes during recent human evolution, since homologous sequences are shown to be present on the X chromosome alone in the chimpanzee and gorilla.     

A Y-chromosomal DNA fragment is conserved in human and chimpanzee.

A human male-specific Y-chromosomal DNA fragment (lambda YH2D6) has been isolated. By deletion-mapping analysis, 2D6 has been localized to the euchromatic portion of the long arm (Yq11) of the human Y chromosome. Among great apes, this fragment was found to be conserved in male chimpanzee but was lacking in male gorilla and male orangutan. No homologous fragments were detected in females of orangutan, gorilla, chimpanzee, or human. Nucleotide sequence analysis indicated the presence of partial-Alu-elements and of sequences similar to the GATA repeats of the snake Bkm sequence.     

Chromosomal localization of Sau3A repetitive DNA revealed by in situ hybridization

The Sau3A DNA family consists of unique alphoid human repetitive DNA which is prone to be excised from the chromosomes and exhibits restriction fragment length polymorphism. We studied the chromosomal localization of the DNA by in situ hybridization using cultured normal human lymphocytes. Under standard hybridization conditions, the sequence hybridized with the centromeric regions of chromosomes 1, 2, 4, 11, 15, 17, 18, 19 and X, but under high stringency hybridization conditions, it hybridized with the centromeric regions of chromosomes 1, 17 and X, and particularly chromosome 11. Based on these results, we discuss the evolutionary relationship among the sequences of the Sau3A DNA family.     

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.     

Extensive sequence homologies between Y and other human chromosomes

Twenty-six human Y-chromosome-derived DNA sequences, free of repetitive material, were used to probe male and female genomic blots. We present data from a detailed analysis and chromosomal location of the bands detected by such probes, which demonstrate extensive DNA sequence homology between the mammalian sex chromosomes and autosomes. Under stringent conditions, nine Y-derived probes reacted exclusively with the Y chromosome, 12 probes detected homologous sequences present on both the Y and the X, four probes detected homologies between Y and autosome(s) without any X counterpart and, finally, one probe hybridized to homologous sequences on Y, X and autosome(s). These data are consistent with the hypothesis of a common evolutionary origin for the mammalian sex chromosomes and reveal structural similarities between Y-located and autosomal non-repetitive sequences.     

Recent Origin of Dioecious and Gynodioecious Y Chromosomes in Papaya

Sex of dioecious and gynodioecious papayas is controlled by two slightly different Y chromosomes, Y for males and Y^h for hermaphrodites. All combinations of the Y and/or Y^h chromosomes are lethal. We investigated the features of paired dioecious X- and Y-specific bacterial artificial chromosomes (BACs) and compared their sequences to corresponding gynodioecious X- and Y-specific BACs. Numerous chromosomal rearrangements were detected between the X- and Y-specific BACs, including inversions, deletions, insertions, and duplications. DNA sequence expansion was documented on the Y BAC. Dioecious and gynodioecious X-specific BACs were virtually identical. The Y- and Y^h-specific BACs shared high degree of DNA sequence identity, but local chromosomal rearrangements were detected, as the consequence of suppression of recombination in the male specific region and the isolation of Y and Y^h chromosomes enforced by the lethal effect. Analysis of sequence divergence between three dioecious X and Y gene pairs resulted in the estimated ages of divergence from 0.6 to 2.5 million years, reinforcing the hypothesis of a recent origin of the papaya sex chromosomes. The estimated age of divergence between Y and Y^h chromosomes was approximately 73,000 years for Gene 5. Our findings indicate that Y and Y^h chromosomes evolved from a common ancestral Y chromosome, possibly prior to the origin of agriculture. The existence of a hermaphrodite Y^h chromosome is less likely to have resulted from human selection as once suggested.     

Analysis of two 47,XXX males reveals X-Y interchange and maternal or paternal nondisjunction

Summary Two cases of 47,XXX males were studied, one of which has been published previously (Bigozzi et al. 1980). Analysis of X-linked restriction fragment length polymorphisms revealed that in this case, one X chromosome was of paternal and two were of maternal origin, whereas in the other case, two X chromosomes were of paternal and one of maternal origin. Southern blot analysis with Y-specific DNA probes demonstrated the presence of Y short arm sequences in both XXX males. In one case, the results obtained pointed to a paracentric inversion on Yp of the patient's father. In situ hybridization indicated that the Y-specific DNA sequences were localized on Xp22.3 in one of the three X chromosomes in both cases. The presence of Y DNA had no effect on random X inactivation. It is concluded that both XXX males originate from aberrant X-Y interchange during paternal meiosis, with coincident nondisjunction of the X chromosome during maternal meiosis in case 1, and during paternal meiosis II in case 2.     

Steroid sulfatase gene in XX males.

The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid methods to visualize Y-specific repeated DNA sequences in cytological preparations

We described rapid methods to detect Y-specific repeated DNA sequences in cytological preparations using in situ hybridization. A human Y chromosome specific DNA probe with an insert equivalent to that in pHY2.1 was labelled with [alpha-32P]dCTP or photobiotin, and hybridized to chromosome preparations. Signals were visualized specifically on Y chromosomes after 1 day's autoradiography or a couple of hours treatment with streptavidin alkaline phosphatase/BCIP/NBT. These methods are useful for molecular confirmation of Y-autosomal translocations.     

Comparative mapping of human alphoid satellite DNA repeat sequences in the great apes

Heterochromatic regions of chromosomes contain highly repetitive, tandemly arranged DNA sequences that undergo very rapid variation compared to unique DNA sequences that are predominantly conserved. In this study the chromosomal basis of speciation has been looked at in terms of repeat sequences. We have hybridized twenty-one chromosome-specific human alphoid satellite DNA probes to metaphase spreads of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) to investigate the evolutionary relationship of heterochromatic regions among such hominoid species. The majority of the probes did not hybridize to their corresponding equivalent chromosome but presented hybridization signals on non-corresponding chromosomes. Such observations suggest that rapid changes may have occurred in the ancestral alphoid satellite DNA sequence, resulting in divergence among the great ape species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Strong linkage disequilibrium between the XY274 polymorphism and the pseudoautosomal boundary.

The pseudoautosomal boundary is defined by an Alu repeat element on the Y chromosome. The Alu element is found on all Y chromosomes and on no X chromosomes, establishing it as part of Y-specific sequences. Distal to the Alu element, sequences from the X and Y are strictly homologous, suggesting that the boundary is formed by an abrupt break in sequence homology. Further investigation of the function of the boundary has been undertaken by examining the population structure of an MspI restriction-site polymorphism (XY274), which is located 274 bp distal to the Alu insertion site. Southern blot and polymerase chain reaction analysis demonstrate fixation of the high allele (noncutting or AT base pair) of XY274 on the Y chromosome in most populations, while a full range of high allele frequencies is found on the X chromosomes of different populations. Two exceptions to fixation on the Y chromosome were found in African populations. The level of linkage disequilibrium suggests that the first few hundred base pairs of the pseudoautosomal region on the Y chromosome share a single common origin more recent than the origin of the species.